Laminins are a family of extracellular matrix proteins localized primarily in the basement membranes which regulate various cellular functions such as adhesion, motility, growth, differentiation, wound healing, and tumor invasion through an interaction with specific integrins or other receptors on the cell surface (Timpl, R., Curr. Opin. Cell Biol., 8: 618, 1996; Howe, A. et al, Curr. Opin. Cell Biol., 10:220, 1998; Colognato, H. and Yurchenco, P. D., Dev. Dyn., 218:213, 2000). Laminins consist of three different subunits (α, β and γ chains) which form the cross-shaped structure linked by disulfide bonds (Ekblom, M. et al., Ann. N. Y Acad. Sci., 857:194, 1998). Thus far, 5α, 3β and 3γ chains have been identified, at least 15 isoforms (laminin 1-15) are formed by various combinations of these subunits (Colognato, H. and Yurchenco, P. D., Dev. Dyn., 218:213, 2000; Burgeson, R. E. et al., Matrix Biol., 14:209, 1994; Miner, J. H. et al., J. Cell Biol., 137:685, 1997). Among these laminin isoforms, laminin-5, an isoform consisting of α3, β3, and γ2 chains, has a unique structure and biological activity. Laminin-5 was originally identified as a keratinocyte-derived matrix protein (Carter, W. G. et al., Cell, 65:599, 1991), but, unlike other isoforms, it lacks some domains found in the N-terminal regions of the three subunits (Aumailley, M. and Rousselle, P., Matrix Biol., 18:19, 1999). The laminin-5 precursor (460 kDa) is processed in keratinocytes. This precursor is secreted, followed by the proteolytic conversion of the α3 chain (200 kDa) and the γ2 chain (155 kDa) to 165 and 105 kDa, respectively (Marinkovich, M. P. et al., J. Biol. Chem., 267:17900, 1992; Rousselle, P. et al., J. Cell Biol., 114:567, 1991).
Laminin-5 regulates the stable adhesion of the epithelium to the underlying connective tissue (Marinkovich, M. P. et al., J. Biol. Chem., 267:17900, 1992; Rousselle, P. et al., J. Cell Biol., 114:567, 1991; Niessen, C. M. et al., Exp. Cell. Res., 211:360, 1994), and influences the cell's behavior by interacting with cell surface receptors, such as integrins α3β1 and α6β4 (Carter, W. G. et al., Cell, 65:599, 1991; Rousselle, P. et al., J. Cell Biol., 114:567, 1991; Niessen, C. M. et al., Exp. Cell. Res., 211:360, 1994; Rousselle, P. and Aumailley, M., J. Cell Biol., 125:205, 1994). Among the integrins expressed in normal human keratinocytes, integrin α3β1 is involved in the formation of focal adhesions, which are associated with actin-containing stress fibers and mediation of keratinocyte motility (DiPersio, C. M. et al., J. Cell Sci., 108:2321, 1995; Zhang, K. and Kramer, R. H., Exp. Cell. Res., 227:309, 1996). Integrin α6β4 forms the hemidesmosome structure of epithelial cells, mediates adhesion, migration, and wound healing of epithelial cells, and is involved in the invasion of carcinoma cells (Niessen, C. M. et al., Exp. Cell. Res., 211:360, 1994; Mercurio, A. M. et al, Curr. Opin. Cell Biol., 13:541, 2001). Mutations in laminin-5 or integrin α6β4 cause a Herlitz-type junctional epidermolysis bullosa, which is characterized by a splitting of the epidermal/dermal junctions (Aberdam, D. et al., Nat. Genet., 6:299, 1994; Korge, B. P. and Krieg, T., J. Mol. Med., 74:59, 1996).
Besides laminin-5, the laminin α3 chain is found in laminin-6(α3β1γ1), laminin-7(α3β2γ1) and laminin-13(α3β2γ3), however the laminin β3 and γ2 chains are found only in laminin-5. The laminin α3 chain contains a C-terminal globular domain that consists of five globular modules LG1-LG5, each approximately 200 amino acid residues in length (Talts, J. F. et al., FEBS Lett., 426:71, 1998; Timple, R. et al., Matrix Biol., 19:309, 2000). Mapping studies using various laminin isoforms have mapped the localization of the integrin-dependent cell adhesion site to the globular domain of laminin α chains (Baker, S. E. et al., J. Cell Sci., 109:2509, 1996; Hirosaki, T. et al., J. Biol. Chem., 275:22495, 2000). The C-terminal LG3 domain in the α3 chain is essential for the unique activity of laminin-5. Recombinant LG3 (rLG3) domain in rats and recombinant laminin-5 proteins serially lacking the LG domains in humans promote cell adhesion and migration by interacting with integrin α3β1 (Hirosaki, T. et al., J. Biol. Chem., 275:22495, 2000; Shang, M. et al., J. Biol. Chem., 276:33045, 2000). Studies on the rLG proteins of the human laminin α3 chain have shown that the LG2 domain contains an integrin α3β1-binding site, while the LG4 and LG5 domains weakly interact with the heparin sulfate proteoglycans (Mizushima, H. et al., Cell Growth Differ., 8:979, 1997). In addition, while the LG4-LG5 fragment of the laminin α3 chain, itself does not exhibit activity, it stimulates cell migration in the presence of the mature laminin-5, which suggests a regulatory role in cell migration (Tsubota, Y. et al., Biochem. Biophys. Res. Commun., 278:614, 2000). However, the direct demonstration of integrin-dependent cell adhesion in the human laminin α3 chain LG3 domain has not been reproduced using rLG domain fragments, possibly due to difficulties in expressing the soluble LG proteins.
Meanwhile, it has been recognized that the primary adhesion site of laminins is located in the C-terminal LG domain region of laminin a chains. Various LG domains of the laminin α chain have been shown to mediate cell adhesion and migration and bind to heparin, α-dystroglycan, syndecans, and integrins (Howe, A. et al., Curr. Opin. Cell Biol., 10:220, 1998). Although it has been demonstrated that the LG3 domain of the human laminin-5 α3 chain is essential for the promotion of cell adhesion and motility by laminin-5 (Hirosaki, T. et al., J. Biol. Chem., 275:22495, 2000), there has not been any further report on the active sites for the biological functions and integrin binding in the LG3 domain.
Accordingly, to clarify the functions of the LG domains of the human laminin-5 α3 chain and to further determine biologically active core sequences, the present inventors prepared the recombinant human laminin-5 α3 chain LG domains expressed in the form of monomeric, soluble fusion proteins and synthetic peptides within the LG3 domain of the laminin-5 α3 chain, and examined their cell adhesion and spreading activities. As a result, the present inventors confirmed that human laminin-5 α3 LG3 domain promoted cell adhesion and spreading activities by acting as a ligand for integrin α3β1, and a motif PPFLMLLKGSTR (1312-1323 residues) of the SEQ ID NO:1 within the LG3 domain, fragments and derivatives thereof are important for cell adhesion and spreading activities as an active site for integrin α3β1 binding, and show excellent cell adhesion activity with Beschitin W microfiber thus can be used in wound care or tissue regeneration, thereby completing the present invention.